To develop preventive interventions for breast cancer (BC), it is important to determine the molecular alterations that characterize the initiating steps of breast tumorigenesis. SAGE (Serial Analysis of Gene Expression) was used to compare the gene expression profiles of normal and DCIS (Ductal Carcinoma In-Situ, a precursor of invasive BC) mammary epithelial cells. This technique identified HIN-1 (High In Normal-1), a novel secreted protein that is highly expressed in normal breast epithelial cells, but lost in >90% of primary pre-invasive and invasive BCs and BC cell lines. The high frequency at which HIN- 1 is inactivated in these early tumors suggests that it acts as a novel tumor suppressor. HIN-1 is also highly expressed in normal lung, pancreas, prostate, and salivary gland, all organs containing branching epithelia. This suggests that HIN-1 may be involved in morphogenesis or differentiation. . The overall objective of the proposed research is to characterize the function of HIN-1 and to determine its role in the initiating steps of early BC. Specific aim 1 is to determine the effect of HIN-1 on cell proliferation. HIN-1 will be introduced into BC cell lines using adenoviral and plasmid vectors or by exposure to purified recombinant. HIN-1 and the effects analyzed using colony growth assays and flow cytometric analysis of cell cycle progression. Specific aim 2 will explore the effects of HIN-1 expression on branching morphogenesis of mammary epithelial cells in a three-dimensional culture system. Specific aim 3 will address the ability of HIN-1 to suppress tumor cell growth in nude mice using BC cell lines transfected with a tetracycline inducible HIN-1 vector. The results of these studies will thus determine the role of HIN-1 as a potential tumor suppressor gene and, since it is inactivated frequently in early BC, may provide an excellent target for preventive intervention.